The long-term goal of our research is to determine the nature of the derangements in endothelial function that occur with CH-induced pulmonary hypertension. Our previous work has established that eNOS expression is selectively elevated in the arterial segments of the pulmonary circulation that demonstrate vascular remodeling and elevated vascular resistance. Our data suggest that this upregulation of eNOS expression is the result of altered mechanical forces associated with remodeling, such as elevated shear stress or intraluminal pressure. It is likely that these stimuli or hypoxia per se could influence endothelial cell Em and calcium influx as well. Indeed, in pilot experiments presented below we document endothelial depolarization and lower [Ca2+]j in intact small pulmonary arteries from CH-exposed rats compared to controls. In addition, we have found that VSMC Em is relatively depolarized in pulmonary arteries from CH rats compared to controls, even when vessels are acutely returned to normoxia. There is evidence in other beds that electrical coupling between VSMCs and endothelial cells exists, such that the endothelial cell Em could be influenced by effects of CH on VSMC Em. In addition, we have evidence that calcium entry pathways may be diminished following CH, which could also contribute to lowered [Ca2+]j. The objective of the present proposal is to determine the effects of CH on pulmonary endothelial calcium homeostasis, testing the central hypothesis that CH exposure leads to endothelial cell depolarization and decreased calcium influx. The planned experiments will establish the factors that constrain activity of calcium-sensitive endothelial pathways such as NO production in this clinically relevant setting. Our laboratory has developed an array of techniques necessary to clearly assess this question, including the measurement of endothelial cell calcium and Em in intact small intrapulmonary arteries from control and CH-exposed animals as well as single cell electrophysiology and calcium imaging methodology. ? ?
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